Project Summary thalassemia is one of the most common genetic disorders worldwide. Homozygous thalassemia major, or Cooley's Anemia (CA), is characterized by the complete absence of the -globin polypeptide chains of hemoglobin. The hypothesis that a human fetal to adult hemoglobin switching cassette can generate an animal model of CA that mimics the disease in humans will be tested in this proposal. This project creates novel preclinical animal models of CA that survive solely on human fetal hemoglobin at birth and are blood transfusion dependent for life upon completion of their hemoglobin switch after birth. These humanized mouse models of CA have the following characteristics: express 100% human hemoglobin in adult red blood cells; have a functional human -globin and mutant human 0-globin knock-in alleles at each murine -globin locus; complete the human - to 0-globin gene switch after birth; synthesize no functional adult -globin chains; are blood transfusion dependent for life after birth; and are genetically heritable. These novel CA mice can be utilized as preclinical animal models for the study of the following NHLBI Division of Blood Diseases and Resources (DBDR) stated goals: regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; studying iron overload in disease; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia. The first main objective and Specific Aim of this project is to generate humanized CA mice that are more amenable for postnatal studies by increasing the newborn levels of fetal hemoglobin (HbF) and/or the minor adult hemoglobin (HbA2). The second main objective and Specific Aim of this proposal is to utilize novel stem and progenitor cell therapies to rescue the CA mice from their lethal anemia. Humanized CA mice will receive pluripotent stem cell derived pure definitive erythroid progenitor cell therapy as a replacement for traditional transfusion therapy. Additionally, allogenic bone marrow transplantation will be used to cure the CA mice in the absence of all toxic conditioning. Lastly, autologous induced pluripotent stem (iPS) cell therapy will be used to cure the humanized CA mice after correction, differentiation, and transplantation back into CA recipients. Successful completion of the goals of this project could have a significant impact upon the clinical treatment of CA patients. Furthermore, these preclinical animal models of CA, once distributed to the scientific community, will be valuable for the study of the regulation of globin gene expression, synthesis, and switching; studies of stress and ineffective erythropoiesis; development of transfusion and iron chelation therapies; studies of iron homeostasis in disease; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell- based therapies for the correction of thalassemia.